U.S. patent application number 10/295820 was filed with the patent office on 2003-06-12 for antagonists of intestinotrophic glp-2 peptides.
This patent application is currently assigned to 1149336 Ontario Inc.. Invention is credited to Crivici, Anna E., Drucker, Daniel J., Sumner-Smith, Martin.
Application Number | 20030109449 10/295820 |
Document ID | / |
Family ID | 24745881 |
Filed Date | 2003-06-12 |
United States Patent
Application |
20030109449 |
Kind Code |
A1 |
Drucker, Daniel J. ; et
al. |
June 12, 2003 |
Antagonists of intestinotrophic GLP-2 peptides
Abstract
Antagonists of glucagon-like peptide 2, have been identified.
Their effects on the growth of gastrointestinal tissue are
described. Its formulation as a pharmaceutical, and its therapeutic
and related uses in treating bowel tissue, are described. Also
described are methods of identifying antagonists of glucagon-like
peptide 2.
Inventors: |
Drucker, Daniel J.;
(Toronto, CA) ; Crivici, Anna E.; (San Diego,
CA) ; Sumner-Smith, Martin; (Bolton, CA) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
1149336 Ontario Inc.
|
Family ID: |
24745881 |
Appl. No.: |
10/295820 |
Filed: |
November 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10295820 |
Nov 18, 2002 |
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09233934 |
Jan 19, 1999 |
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6489295 |
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10295820 |
Nov 18, 2002 |
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08683890 |
Jul 19, 1996 |
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5994500 |
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Current U.S.
Class: |
514/11.7 ;
435/7.23; 514/19.3; 530/324 |
Current CPC
Class: |
G01N 2333/605 20130101;
A61P 1/12 20180101; A61P 43/00 20180101; Y02A 50/471 20180101; A61K
38/00 20130101; A61P 3/04 20180101; A61P 31/00 20180101; Y02A 50/30
20180101; C07K 14/605 20130101; A61P 35/00 20180101; A61P 1/00
20180101 |
Class at
Publication: |
514/12 ; 530/324;
435/7.23 |
International
Class: |
A61K 038/17; C07K
014/435; C07K 007/08; G01N 033/574 |
Claims
What is claimed is:
1. A polypeptide which comprises an amino acid sequence
corresponding to that of a first reference mammalian GLP-2, which
polypeptide is mutated so that: (i) from one to four of any of the
first four N-terminal residues are deleted; or (ii) at least one
amino acid selected from the amino acid positions corresponding to
the amino acid positions of human GLP-2 at Asp.sup.15, Phe.sup.22,
Thr.sup.29, Thr.sup.32 and Asp.sup.33 is substituted with an amino
acid which does not naturally occur at that position in the
reference GLP-2; or (iii) position Ala.sup.2 is substituted with an
amino acid selected from the group consisting of Leu, Cys, Glu,
Arg, Trp, and PO.sub.3-Tyr.sup.2; or (iv) a combination of (i),
(ii), and/or (iii) is mutated; said polypeptide exhibiting GLP-2
antagonist activity.
2. A peptide as defined in claim 1, wherein the reference mammalian
GLP-2 is selected from the group consisting of human GLP-2, degu
GLP-2, ox GLP-2, porcine GLP-2, guinea pig GLP-2 and hamster
GLP-2.
3. A peptide as defined in claim 1, wherein the reference mammalian
GLP-2 is human GLP-2.
4. A peptide as defined in claim 1, said peptide having a
substitution at a position selected from the group consisting of
Asp.sup.15, Phe.sup.22, Thr.sup.29, Thr.sup.32, and Asp.sup.33.
5. A peptide as defined in claim 1, said peptide selected from the
group consisting of [Leu.sup.2]GLP-2, [Glu.sup.2]GLP-2,
[Arg.sup.2]GLP-2, [Trp.sup.2]GLP-2, [PO.sub.3-Tyr.sup.2]GLP-2, and
[Cys.sup.2]GLP-2.
6. A peptide as defined in claim 1, which is GLP-2(2-33).
7. A peptide as defined in claim 1, which is GLP-2(3-33).
8. A peptide as defined in claim 1, which is GLP-2(4-33).
9. A peptide as defined in claim 1, which is GLP-2(5-33).
10. A peptide as defined in claim 1, which peptide further
comprises one or more substitutions selected from the group
consisting of: (i) substitution of Ala.sup.2 with Val, Gly, or
D-Ala; (ii) substitution of Met.sup.10 with Leu, Ile, Nle, or Ala;
(iii) an amino terminal blocking group; and (iv) a carboxy terminal
blocking group.
11. A peptide as defined in claim 10, selected from the group
consisting of: [Gly.sup.2, Ala.sup.15]GLP-2; [Gly.sup.2,
Ala.sup.22]GLP-2; [Gly.sup.2, Ala.sup.29]GLP-2; [Gly.sup.2,
Ala.sup.32]GLP-2; and [Gly.sup.2, Ala.sup.33]GLP-2.
12. A pharmaceutical composition comprising a therapeutically
effective amount of a peptide according to claim 1, and a
pharmaceutically acceptable carrier.
13. A method for suppressing growth of small bowel tissue in a
subject, comprising the step of administering to the subject an
effective amount of a funtional antagonist of GLP-2.
14. A method of reducing hyperplasia or inducing hypoplasia of
small bowel tissue in a subject, comprising the step of delivering
to the subject a therapeutically effective amount of a peptide
according to claim 1, which results in the reduction of hyperplasia
or induction of hypoplasia of small bowel tissue.
15. The method of claim 14, wherein the subject suffers from
obesity or the subject requires bowel rest prior to treatment with
chemotherapy or radiotherapy.
16. A method of treating a gastrointestinal disease, wherein the
method comprises administering to a subject having the
gastrointestinal disease a therapeutically effective amount of a
peptide according to claim 1, which results in the amelioration of
a pathological effect or symptom of the gastrointestinal
disease.
17. The method of claim 16, wherein the gastrointestinal disease is
selected from the group consisting of small bowel cancer, cholera,
irritable bowel, bowel motility disorders, and chronic
diarrhea.
18. A method of identifying a GLP-2 antagonist comprising the steps
of: (1) obtaining a GLP-2 analog having a structural alteration
selected from (i) a deletion of at least one amino acid, and (ii) a
substitution of at least one amino acid position with an amino acid
which does not naturally occur at that position, and (iii) a
combination of (i) and (ii); (2) treating a mammal with said analog
using a regimen capable of eliciting a decrease in small bowel
weight; and (3) determining the effect of said analog on small
bowel weight relative to a control mammal receiving vehicle only,
whereby said functional antagonist of GLP-2 is identified as an
analog which elicits a decrease in said small bowel weight.
19. The method as defined in claim 18, wherein the GLP-2 analog
structural alteration is a deletion of from one to four of any of
the first four N-terminal residues.
20. The method as defined in claim 18, wherein the GLP-2 analog
structural alteration is a substitution at a position selected from
Asp.sup.15, Phe.sup.22, Thr.sup.29, Thr.sup.32, and Asp.sup.33.
21. A GLP-2 antagonist identified by the method of claim 18.
22. A method of identifying GLP-2 antagonists comprising the steps
of: (1) obtaining a GLP-2 analog having a structural alteration
selected from (i) a deletion of at least one amino acid, and (ii) a
substitution of at least one amino acid position with an amino acid
which does not naturally occur at that position, and (iii) a
combination of (i) and (ii); (2) treating a mammal with said analog
co-administered with GLP-2 or an intestinotrophic analog of GLP-2,
using a regimen capable of eliciting a reduction in the small bowel
weight increase seen when GLP-2 is administered alone; and (3)
determining the effect of said analog on small bowel weight
relative to a control animal, receiving GLP-2 alone or a
intestinotrophic analog of GLP-2 alone, whereby said functional
antagonist is identified as an analog which elicits a reduction in
the increase in said bowel weight.
23. The method as defined in claim 22, wherein the GLP-2 analog
structural alteration is a deletion of from one to four of any of
the first four N-terminal residues.
24. The method as defined in claim 22, wherein the GLP-2 analog
structural alteration is a substitution at a position selected from
Asp.sup.15, Phe.sup.22, Thr.sup.29, Thr.sup.32, and Asp.sup.33.
25. A GLP-2 antagonist identified by the method of claim 22.
Description
I. FIELD OF THE INVENTION
[0001] This invention relates to glucagon-related peptides which
are functional antagonists of glucagon-like peptides-2, and to
their use therapeutically to counter hyperplasia or induce
hypoplasia particularly in intestinal tissue.
II. BACKGROUND TO THE INVENTION
[0002] Expression of the glucagon gene yields a tissue-determined
variety of peptide products that are processed from the 160 residue
proglucagon product. The organization of these peptides within the
proglucagon precursor was elucidated by the molecular cloning of
preproglucagon cDNAs from the anglerfish, rat, hamster and bovine
pancreas. These analyses revealed that preproglucagon contains not
only the sequence of glucagon and glicentin, but also two
additional glucagon-like peptides (GLP-1 and GLP-2) separated from
glucagon and each other by two spacer or intervening peptides (IP-I
and IP-II). These peptides are flanked by pairs of basic amino
acids, characteristic of classic prohormone cleavage sites,
suggesting they might be liberated after posttranslational
processing of proglucagon (Drucker, Pancreas, 1990, 5(4):484).
Analysis of the peptides liberated from proglucagon in the
pancreatic islets of Langerhans, for instance, suggests the primary
pancreatic peptide liberated is the 29-mer glucagon, whereas
glicentin, oxyntomodulin, IP-II and the glucagon-like peptides are
more prevalent in the small and large intestines. This
demonstration that the glucagon-like peptides are found in the
intestine has prompted research into the precise structure and
putative function(s) of these newly discovered gut peptides. Most
studies have focussed on GLP-1, because several lines of evidence
suggested that GLP-1 may be an important new regulatory peptide.
Indeed, it has been determined that GLP-1 is the most potent known
peptidergic stimulus for insulin release, an action mediated in a
glucose-dependent manner through interaction with receptors on
pancreatic .beta. cells. GLP-1 and its derivatives are in
development for use in the treatment of diabetics.
[0003] With respect to the biological role of GLP-2, co-pending
U.S. application Ser. No. 08/422,540 (PCT Publ. No. WO 96/32414),
incorporated in its entirety herein by reference, discloses that
mammalian GLP-2 acts as a trophic agent, to promote growth of
intestinal tissue. The effect of GLP-2 is marked particularly by
increased growth of the small intestine. Furthermore, co-pending
U.S. application Ser. No. 08/631,273 and PCT Application No. PCT/CA
97/00252, both of which are incorporated in its entirety herein by
reference, disclose that analogs of vertebrate GLP-2 can have
enhanced intestinotrophic activity.
III. SUMMARY OF THE INVENTION
[0004] It has now been discovered that alteration of GLP-2 peptide
structure can yield peptides capable of inhibiting the
intestinotrophic activity of GLP-2. More particularly, and
according to one aspect of the invention, there are provided
antagonists comprising an amino acid sequence corresponding to that
of a first reference mammalian GLP-2 which has been mutated so that
from one to four of any of the first four N-terminal residues are
deleted. In another aspect of the invention, the antagonists
correspond to a reference mammalian GLP-2 that has been mutated so
that at least one amino acid selected from the amino acid positions
corresponding to the amino acid positions of human GLP-2 at
Asp.sup.15, Phe.sup.22, Thr.sup.29, Thr.sup.32 and Asp.sup.33 is
substituted with an amino acid which does not naturally occur at
that position in the reference GLP-2. In another aspect of the
invention, position Ala.sup.2 is substituted with an amino acid
selected from the group consisting of Leu, Cys, Glu, Arg, Trp, and
PO.sub.3-Tyr.sup.2. In yet another aspect of the invention, the
antagonist corresponds to a polypeptide with any combination of the
above substitutions and deletions mutated relative to the reference
mammalian GLP-2.
[0005] Also provided as an aspect of the invention are methods of
producing and identifying GLP-2 antagonists.
[0006] For use in medical or veterinary treatment, there is further
provided by the present invention a pharmaceutical or veterinary
composition comprising an amount of a GLP-2 antagonist effective to
antagonize GLP-2 activity in vivo, and a pharmaceutically or
veterinarily acceptable carrier.
[0007] The GLP-2 antagonist activity of the present GLP-2
antagonists is manifest in vivo as a reduction in the mass of small
bowel tissue or as an ability to inhibit the intestinotrophic
activity of GLP-2 or intestinotrophic analogs thereof. Accordingly,
there is provided, in another aspect of the invention, a method for
reducing the mass or suppressing the proliferation of small bowel
tissue in a subject, including an animal or a human, which
comprises the step of delivering to that subject an amount of a
GLP-2 antagonist of the invention effective to cause a reduction in
the mass of small bowel tissue.
[0008] Subjects for whom such treatment would be useful include
those suffering from hyperplastic conditions of the small
intestine, for example, as a result of GLP-2 overdose or of GLP-2
overproducing tumors, and conditions wherein prophylactic
inducement of small bowel hypoplasia would be useful, for example,
in the treatment of clinical obesity as a non-surgical alternative
to resection of the small intestine.
IV. DETAILED DESCRIPTION OF THE INVENTION
[0009] The present invention relates to therapeutic and related
uses of a novel class of GLP-2 antagonists, particularly for
decreasing the growth rate of gastrointestinal tissue, most
particularly small bowel. The biological effect of the present
GLP-2 antagonists manifests as a decrease in small bowel weight,
relative to a mock treated control or as an ability to inhibit the
intestinotrophic activity of GLP-2 or an intestinotrophic analog of
GLP-2, relative to a control animal given either GLP-2 or an
intestinotrophic analog of GLP-2 alone.
[0010] The present GLP-2 antagonists are structural analogs of the
intestinotrophic GLP-2 peptides. GLP-2 peptides refers collectively
to the various vertebrate forms of GLP-2 and to modified forms
(characterized by at least one addition, deletion, substitution,
and/or incorporation of an amino acid residue with a blocking
group) of the GLP-2 analogs which still retain intestinotrophic
activity. However, as described herein, certain site-specific
alterations of these intestinotrophic GLP-2 peptides can confer
antagonist activity to the site-specifically altered analog.
[0011] Without being limited by following explanation, it is
believed that the site specific alterations which confer antagonist
activity interfere with one of the functional activities of the
GLP-2 hormone peptide, but not all functional activities. For
example, an alteration conferring antagonist activity to a GLP-2
analog may be one which does not inhibit hormone binding to its
cognate receptor, but does prevent the subsequent signal
transduction through the bound receptor. For example, the site
specific alteration of the hormone may prevent dimerization of the
hormone receptor which is necessary to transmit a signal to the
interior of the cell. Such a mechanism for antagonistic activity
has been observed with other hormones such as, for example, human
growth hormone (see Fuh et al., Science, 1992, 256:1677-1680).
[0012] Generally, sites which are highly conserved among mammalian
GLP-2's are candidates for modification in order to obtain an
antagonist. Among mammals, at least residues 1-5, 7, 15, and 22, 29
and 32-33 are highly conserved. Therefore, deletion or substitution
of the residues at these sites can result in a GLP-2 antagonist.
Additionally, certain modifications of sites near these conserved
sites may also cause antagonist activity by disrupting local
tertiary amino acid structure or placement of the adjacent
conserved residues.
[0013] The GLP-2 antagonists of the invention include peptide
derivatives with a sequence derived from a vertebrate GLP-2 in that
one or more of any of the first four N-terminal amino acids
(relative to the sequence of human GLP-2) are deleted. These
analogs are referred to herein as the deletion class of GLP-2
antagonists. From the deletion class of GLP-2 antagonists, it will
be appreciated that GLP-2 antagonism can result from disruption of
the N-terminal structure of GLP-2 within the first four amino
acids. Thus, the deletion class of GLP-2 antagonists comprises:
GLP-2(2-33), GLP-2(3-33), GLP-2(4-33) and GLP-2(5-33),
[desAla.sup.2]GLP-2, [desAsp.sup.3]GLP-2 and
[desGly.sup.4]GLP-2.
[0014] Additionally, the GLP-2 antagonists of the invention include
substitution derivatives of vertebrate GLP-2's. The substitution
class of GLP-2 antagonists includes those antagonists which replace
one of the following amino acids at the following positions
(relative to sequence of the human GLP-2) with another amino acid:
residues 15, 22, 29, 32, and 33. Also included in the substitution
class are those incorporating certain Ala.sup.2 substitutions.
[0015] It is to be understood that, in embodiments of the
invention, the GLP-2 antagonists may incorporate any combination of
a deletion and a substitution, or may incorporate two or more
substitutions at the sites noted.
[0016] A. GLP-2 Antagonists
[0017] The GLP-2 antagonists may accordingly be analogs of human
GLP-2, which has the following sequence:
1 His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn- 1 5 10
Thr-Ile-Leu-Asp-Asn-Leu-Al- a-Ala-Arg-Asp-Phe- 15 20
Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp. 25 30 33
[0018] Unless otherwise specified, the term "GLP-2" refers to the
sequence of human GLP-2.
[0019] The antagonists of the invention are polypeptides which
comprise amino acid sequences corresponding to that of a first
reference mammalian GLP-2 which has been mutated so that:
[0020] (i) from one to four of any of the first four N-terminal
residues are deleted; or
[0021] (ii) at least one amino acid selected from the amino acid
positions corresponding to the amino acid positions of human GLP-2
at Asp.sup.15, Phe.sup.22, Thr.sup.29, Thr.sup.32 and Asp.sup.33 is
substituted with an amino acid which does not naturally occur at
that position in the reference GLP-2; or
[0022] (iii) position Ala.sup.2 is substituted with an amino acid
selected from the group consisting of Leu, Cys, Glu, Arg, Trp, and
PO.sub.3-Tyr.sup.2; or
[0023] (iv) a combination of (i) and (ii), or (ii) and (iii) is
mutated.
[0024] In specific embodiments of the invention, for example, the
GLP-2 antagonists of the invention which are altered at residue
positions 1, 2, 3, 4, 22, 29, 32, and/or 33 may be derivatives of
rat GLP-2 which is an Ala.sup.19 variant of human GLP-2; degu
GLP-2, ox GLP-2, porcine GLP-2, guinea pig GLP-2 and hamster GLP-2,
the sequences of which have been reported by many authors including
Buhl et al in J. Biol. Chem., 1988, 263(18):8621.
[0025] GLP-2 residues which occur at a specific position are
determined by aligning the sequences of GLP-2's isolated from
different vertebrate species and comparing the sequence to the
human sequence, reproduced above.
[0026] Further, the GLP-2 antagonists of the invention which are
altered at residue positions 1, 2, 3, 4, 22, 29, 32, and/or 33 may
be derivatives of GLP-2 agonists such as are described in
co-pending U.S. application Ser. Nos. 08/632,533 and 08/631,273,
and PCT Publication No. WO 96/32414 and PCT Application No. PCT/CA
97/00252.
[0027] Amino acids substitutions appropriate at these sites to
yield an antagonist can readily be determined using the murine
model of GLP-2 antagonism herein described. That is, a GLP-2
compound incorporating a structural alteration is obtained and then
screened in the murine model exemplified herein for GLP-2
antagonism activity. Those GLP-2 compounds which elicit a decrease
in bowel growth and/or inhibit the intestinotrophic activity of
GLP-2 or a GLP-2 agonist, are identified in this screen as GLP-2
antagonists.
[0028] GLP-2 antagonists of the present invention are considered to
be functional antagonists of GLP-2 if, when assessed in the murine
model exemplified herein, the antagonist: (1) consistently mediates
a measurable decrease in small bowel weight relative to a control
animal receiving vehicle alone; and/or (2) when assessed by
co-administration in said murine model with GLP-2 or a GLP-2
agonist (in a molar excess ratio of preferably 10:1, and more
preferably 4:1 over agonist) results consistently in a measurable
inhibition of the intestinotrophic effect of GLP-2 or the GLP-2
agonist, as revealed by a reduction in the increase in small bowel
weight induced by GLP-2 administered alone.
[0029] Particularly suitable for therapeutic use are those
functional antagonists of GLP-2 which mediate a bowel weight
decrease of at least about 10% relative to a control animal
receiving vehicle alone; preferred for therapeutic use are those
which mediate a decrease in small bowel weight of at least 15% or
more.
[0030] The small intestine mass reducing activity of the present
GLP-2 antagonists is noted most significantly in relation to the
jejunum, and particularly the proximal jejunum, and is also noted
in the distal ileum. Additionally, the activity of GLP-2
antagonists may also be noted as a reduction in the crypt/villus
height of the small intestine.
[0031] Alternatively, GLP-2 antagonists can be assessed using the
co-administration model detailed above. In this case, antagonists
are considered to be useful antagonists of GLP-2 if, when
co-administered with GLP-2, or an intestinotrophic analog thereof,
at a molar ratio of about 10:1, or more preferably at a molar ratio
of about 4:1, they diminish the activity of GLP-2 or an
intestinotrophic analog thereof by at least 10%, as manifest by a
reduction in the increase in small bowel weight relative to a
control animal treated with either GLP-2 or the GLP-2 agonist
alone.
[0032] In another aspect of the invention, there is provided a
method useful to identify antagonists of GLP-2, such as those
described above, comprising the steps of:
[0033] 1) obtaining a GLP-2 analog that incorporates an alteration
within the peptide sequence:
[0034] 2) treating a mammal with said analog using a regimen
capable of eliciting a measurable loss of the mass of the
intestine; and
[0035] 3) determining the effect of said analog on small bowel
weight and/or on the crypt/villus height of the crypt cells of the
small intestine relative to a mock treated control animal, whereby
a functional GLP-2 antagonist is identified as an analog of GLP-2
which elicits a decrease in said weight and/or said height.
[0036] In a related aspect of the invention, there is provided
another method useful to identify functional GLP-2 antagonists
comprising the steps of:
[0037] 1) obtaining a GLP-2 analog which incorporates an alteration
within the peptide sequence;
[0038] 2) treating a mammal with said analog in a regimen capable
of inhibiting the intestinotrophic activity of GLP-2 or a GLP-2
agonist; and
[0039] 3) determining the effect of said analog on small bowel
weight and/or on the crypt/villus height of the crypt cells of the
small intestine relative to a control animal given GLP-2 or a GLP-2
agonist, whereby said functional GLP-2 antagonist is identified as
an analog of GLP-2 which inhibits the intestinotrophic activity of
GLP-2 and/or the intestinotrophic activity of a GLP-2 agonist.
[0040] In a preferred version of the methods described above useful
to identify functional GLP-2 antagonists, the GLP-2 analog is
chosen from the GLP-2 antagonists described herein.
[0041] B. Choice of Substituting Amino Acids
[0042] The substituting amino acids can be chosen from the wide
variety of amino acids available to peptide chemists, and include
the D-amino acids as well as the L-amino acids and their numerous
derivatives. Most practically, chosen amino acids will be amenable
to incorporation by solid phase or solution phase synthesis, or by
recombinant DNA production means.
[0043] In a first screen, analogs which are candidates for
antagonistic activity are identified by alanine scanning
mutagenesis or other systematic mutagenesis method. These alanine
substitutions are tested for antagonistic activity using the
methods described in detail herein.
[0044] In another aspect of the invention, more effective GLP-2
antagonists may then be made by drastically changing the character
of the naturally occuring amino acid residue that is important in
forming structural interactions (hydrogen bonding, salt bridging,
hydrophobic interactions, positioning of residues) of the GLP-2
hormone with its target molecule (e.g. receptor). With this goal in
mind, it is not normally necessary to screen each site with
replacements by all 18 of the other naturally occurring residues.
Instead, representative members of residue groups are selected.
Generally, these groups are:
[0045] a. positively charged residues: His, Arg and Lys
[0046] b. negatively charged residues: Asp and Glu
[0047] c. amides: Asn and Gln
[0048] d. aromatic residues: Phe, Tyr, Trp.
[0049] e. hydrophobic residues: Ala, Pro, Gly, Val, Leu, Ile, and
Met
[0050] f. uncharged hydrophilic residues: Ser and Thr.
[0051] When preparing these antagonist candidates, one would choose
a residue from a group other than the type of residue which is
naturally occuring at that position. Extreme substitutions are
generated by selecting a residue from a group with opposed
combinations of features. For example, a negatively charged residue
may be substituted by a positively charged residue.
[0052] In the case of the Ala.sup.2 substitutions, the substituting
amino acids are selected carefully so that antagonists of GLP-2
activity result. It should be noted that substitutions at position
2 can have the effect of enhancing the intestinotrophic activity of
GLP-2. For instance, when Ala.sup.2 is replaced by Gly, the result
is dramatically enhanced intestinotrophic activity as well as
resistance to digestion of the GLP-2 peptide by DPP-IV enzyme. The
GLP-2 antagonists of the present invention can surprisingly also be
generated by substituting Ala.sup.2. In embodiments of the
invention, substituting amino acids at position 2 that are useful
to generate GLP-2 antagonists are selected from Leu, Cys, Glu, Arg,
Trp and PO.sub.3-Tyr. GLP-2 antagonists incorporating these
substitutions have the added advantage that they render the peptide
resistant to digestion by DPP-IV enzyme. Preferably, the Ala.sup.2
substituting amino acid is selected from Cys, Glu, Leu, and
Arg.
[0053] Amino acids substituting for Asp.sup.15, Phe.sup.22,
Thr.sup.29, Thr.sup.32, and Asp.sup.33 are desirably selected from
those incorporating a small hydrophobic side chain, such as Ala,
Gly and Val.
[0054] In embodiments of the invention, the substitution class of
GLP-2 antagonists includes: [Gly.sup.2, Ala.sup.15]GLP-2,
[Ala.sup.15]GLP-2, [Ala.sup.15]GLP-2(2-33),
[Ala.sup.15]GLP-2(3-33), [Ala.sup.15]GLP-2(4-33)
[Ala.sup.15]GLP-2(5-33), [Gly.sup.2, Ala.sup.22]GLP-2, [Gly.sup.2,
Ala.sup.29]GLP-2, [Gly.sup.2, Ala.sup.32]GLP-2, [Gly.sup.2,
Ala.sup.33]GLP-2, [Leu.sup.2]GLP-2, [Glu.sup.2]GLP-2,
[Arg.sup.2]GLP-2, [Trp.sup.2]GLP-2, [PO.sub.3-Tyr.sup.2]GLP-2,
[Cys.sup.2]GLP-2, [Ala.sup.15]GLP-2, [Ala.sup.29]GLP-2,
[Ala.sup.32]GLP-2 and [Ala.sup.33]GLP-2.
[0055] C. Additional Modifications for Improving the Properties of
the Analogs of the Invention
[0056] The present GLP-2 antagonists, while incorporating a
structural alteration of the type noted, may have various amino
acid sequences consistent with the sequences of GLP-2 per se or of
GLP-2 agonists. The GLP-2 antagonists may also be analogs of
vertebrate GLP-2 agonists, in which collateral modifications have
been made to enhance other biochemical, biological or physiological
properties of the peptide. Such modifications include, for example
(in those peptides for which antagonism is conferred by
substitution other than at position 2), the substitution of native
Ala.sup.2 by an amino acid that renders the GLP-2 antagonist
resistant to digestion by the enzyme DPP-IV. An amino acid suitable
for this purpose includes particularly Gly. Also, the Met.sup.10
residue can be replaced by an oxidatively more stable amino acid,
such as Leu, Nle, Ile or Ala. Such Met.sup.10-substituted analogs
are accordingly more stable during synthesis, work-up, and storage.
Another modification in this context is replacement of the amino
acid at position 20 by an amino acid other than Arg. In certain
applications, particularly for the synthetic generation of
pharmaceutically or veterinarily acceptable peptides, this
modification is desirable to avoid the retention by the Arg residue
of counterions from solvents such as TFA.
[0057] Within the scope of the present invention are also molecules
in which the N- or C-terminus has been modified to incorporate a
blocking group of the type used conventionally in the art of
peptide chemistry to protect peptide termini from undesired
biochemical attack and degradation in vivo.
[0058] Suitable N-terminal protecting groups include, for example,
C.sub.1-5alkanoyl groups such as acetyl. Also suitable as
N-terminal protecting groups are amino acid analogs lacking the
amino function. Suitable C-terminal protecting groups include
groups which form ketones or amides at the carbon atom of the
C-terminal carboxyl, or groups which form esters at the oxygen atom
of the carboxyl. Ketone and ester-forming groups include alkyl
groups, particularly branched or unbranched C.sub.1-5alkyl groups,
e.g., methyl, ethyl and propyl groups, while amide-forming groups
include amino functions such as primary amine, or alkylamino
functions, e.g., mono-C.sub.1-5alkylamino and
di-C.sub.1-5-alkylamino groups such as methylamino, ethylamino,
dimethylamino, diethylamino, methylethylamino and the like. Amino
acid analogs are also suitable for protecting the C-terminal end of
the present compounds, for example, decarboxylated amino acid
analogs such as agmatine.
[0059] Embodiments of the invention specifically include such
analogs in which the N-terminal blocking group is acetyl; and
analogs in which the C-terminal blocking group is an amine, e.g.,
--NH.sub.2.
[0060] D. Synthesis of the GLP-2 Antagonists
[0061] The present GLP-2 antagonists can be synthesized using
standard techniques of peptide chemistry and can be assessed for
GLP-2 antagonist activity, all according to the guidance provided
herein. Those GLP-2 antagonists that incorporate only L-amino acids
can be produced in commercial quantities by application of
recombinant DNA technology. For this purpose, DNA coding for the
desired GLP-2 antagonist is incorporated into an expression vector
and transformed into a microbial, e.g., yeast, or other cellular
host, which is then cultured under conditions appropriate for GLP-2
antagonist expression. A variety of gene expression systems have
been adapted for this purpose, and typically drive expression of
the desired gene from expression regulatory elements used naturally
by the chosen host. Because GLP-2 does not require post
translational glycosylation for its activity, its production may
conveniently be achieved in bacterial hosts such as E. coli. For
such production, DNA coding for the selected GLP-2 antagonist may
usefully be placed under expression controls of the lac, trp or PL
genes of E. coli. As an alternative to expression of DNA coding for
the GLP-2 antagonist per se, the host can be adapted to express
GLP-2 antagonist as a fusion protein in which the GLP-2 antagonist
is linked releasably to a carrier protein that facilitates
isolation and stability of the expression product.
[0062] In an approach universally applicable to the production of
selected GLP-2 antagonists, and one used necessarily to produce
GLP-2 antagonist forms that incorporate non-genetically encoded
amino acids and N- and C-terminally derivatized forms, the well
established techniques of automated peptide synthesis are employed,
general descriptions of which appear, for example, in J. M. Stewart
and J. D. Young, Solid Phase Peptide Synthesis, 2nd Edition, 1984,
Pierce Chemical Company, Rockford, Ill.; and in M. Bodanszky and A.
Bodanszky, The Practice of Peptide Synthesis, 1984,
Springer-Verlag, New York; Applied Biosystems 430A Users Manual,
1987, ABI Inc., Foster City, Calif. In these techniques, the GLP-2
antagonist is grown from its C-terminal, resin-conjugated residue
by the sequential addition of appropriately protected amino acids,
using either the Fmoc or tBoc protocols, as described for instance
by Orskov et al, 1989, supra.
[0063] For the incorporation of N- and/or C-protecting groups
protocols conventional to solid phase peptide synthesis methods can
also be applied. For incorporation of C-terminal protecting groups,
for example, synthesis of the desired peptide is typically
performed using, as solid phase, a supporting resin that has been
chemically modified so that cleavage from the resin results in a
peptide having the desired C-terminal protecting group. To provide
peptides in which the C-terminus bears a primary amino protecting
group, for instance, synthesis is performed using a
p-methylbenzhydrylamine (MBHA) resin so that, when peptide
synthesis is completed, treatment with hydrofluoric acid releases
the desired C-terminally aminated peptide. Similarly, incorporation
of an N-methylamine protecting group at the C-terminus is achieved
using N-methylaminoethyl-derivatized DVB resin, which upon HF
treatment releases peptide bearing an N-methylamidated C-terminus.
Protection of the C-terminus by esterification can also be achieved
using conventional procedures. This entails use of resin/blocking
group combination that permits release of side-chain protected
peptide from the resin, to allow for subsequent reaction with the
desired alcohol, to form the ester function. FMOC protecting
groups, in combination with DVB resin derivatized with
methoxyalkoxybenzyl alcohol or equivalent linker, can be used for
this purpose, with cleavage from the support being effected by TFA
in dichloromethane. Esterification of the suitably activated
carboxyl function e.g. with DCC, can then proceed by addition of
the desired alcohol, followed by deprotection and isolation of the
esterified peptide product.
[0064] Incorporation of N-terminal protecting groups can be
achieved while the synthesized peptide is still attached to the
resin, for instance by treatment with suitable anhydride and
nitrile. To incorporate an acetyl protecting group at the
N-terminus, for instance, the resin-coupled peptide can be treated
with 20% acetic anhydride in acetonitrile. The N-protected peptide
product can then be cleaved from the resin, deprotected and
subsequently isolated.
[0065] Once the desired peptide sequence has been synthesized,
cleaved from the resin and fully deprotected, the peptide is then
purified to ensure the recovery of a single oligopeptide having the
selected amino acid sequence. Purification can be achieved using
any of the standard approaches, which include reversed-phase
high-pressure liquid chromatography (RP-HPLC) on alkylated silica
columns, e.g. C.sub.4-, C.sub.8-, or C.sub.18-silica. Such column
fractionation is generally accomplished by running linear
gradients, e.g. 10-90%, of increasing % organic solvent, e.g.
acetonitrile, in aqueous buffer, usually containing a small amount
(e.g. 0.1%) of pairing agent such as TFA or TEA. Alternatively,
ion-exchange HPLC can be employed to separate peptide species on
the basis of their charge characteristics. Column fractions are
collected, and those containing peptide of the desired/required
purity are optionally pooled. In one embodiment of the invention,
the peptide is then treated in the established manner to exchange
the cleavage acid (e.g. TFA) with a pharmaceutically or
veterinarily acceptable acid, such as acetic, hydrochloric,
phosphoric, maleic, tartaric, succinic and the like, to provide a
water soluble salt of the peptide.
[0066] E. Uses of the GLP-2 Antagonists of the Invention
[0067] According to the present invention, the GLP-2 antagonist is
administered to treat subjects, including animals and humans, that
would benefit from decreased gastrointestinal tissue growth rate.
In one aspect, subject candidates are those who would benefit from
decreased mass of small intestine tissue. The effects of GLP-2
antagonists on this tissue, as evidenced by the results exemplified
herein, is dramatic and would clearly benefit those subjects
suffering from diseases or conditions marked by hyperplasia in the
small intestinal tract mucosa, which include GLP-2 producing
tumors. Another group of subjects who would clearly benefit from
the effects of GLP-2 antagonists are those in whom it would be
useful to induce hypoplasia of small intestine tissue, for example,
subjects who will in the near future be receiving radiotherapy or
chemotherapy or subjects who are receiving radiotherapy or
chemotherapy. Small intestine epithelial cells are characterized by
rapid cell division and are thus particularly susceptible to damage
by radiotherapy or chemotherapy. Indeed, cell damage to the small
intestinal epithelial cells is the cause of significant mortality
and morbidity in cancer subjects undergoing therapy. Thus, it would
be desirable to slow the growth rate of these cells immediately
prior to initiation of these therapies and during the course of the
treatment. The ability to decrease the growth rate of small
intestine cells in these subjects, and thus achieve bowel rest
prior to treatment with chemotherapy or radiotherapy, would have
the additional benefit of allowing higher doses of radiotherapeutic
and chemotherapeutic agents.
[0068] Another clinical situation wherein a functional antagonist
of GLP-2 would be clinically useful is the treatment of a subject,
including an animal or a human, who has been chronically or acutely
overdosed with GLP-2 or a GLP-2 agonist. Yet another potential
application of functional antagonists of GLP-2 is to block
transport of toxins or other drugs across the mucosal layer.
Pathogenic effects in some diseases arise as a result of absorption
of toxins or drugs via the intestinal epithelium. Elimination of
the absorptive capacity of the small bowel by reducing the
intestinal epithelium may be beneficial. For example, some diseases
such as cholera are lethal because cholera toxin binds to receptors
in the intestinal epithelium itself, leading to dehydration and
death. GLP-2 antagonists may produce bowel rest, eliminating the
target tissue for the toxin (intestinal epithelium) and hence the
pathological response to cholera.
[0069] Yet another group of subjects who would benefit from a
decrease in the mass of the small intestine are those suffering
from obesity, as a alternative to surgical intervention such as
resection of the small intestine. Thus, in one aspect the invention
provides a method for causing a decrease in the proliferation of
small bowel tissue in a subject in need thereof, comprising the
step of delivering to the subject an amount of a GLP-2 antagonist
of the present invention effective to antagonize GLP-2. The
therapeutic efficacy of the GLP-2 antagonist treatment may be
monitored by enteric biopsy to examine the villus morphology or by
biochemical assessment of nutrient absorption. Additionally,
efficacy may be assessed using a clinical endpoint relevant to the
particular condition treated, for example, weight loss. In a
related aspect the invention provides a method of treating a
subject suffering from a gastrointestinal disease, by administering
a therapeutically effective amount of a functional antagonist of
the present invention, together with a pharmaceutically or
veterinarily acceptable carrier, in order to reduce a pathological
symptom of the gastrointestinal disease. For example, subjects with
small bowel cancer may be administered GLP-2 antagonist to decrease
the size of the tumor. Alternatively, subjects with bowel motility
disorders, irritable bowel, and chronic diarrhea may benefit from
GLP-2 antagonist to increase motility and/or reduce diarrhea.
[0070] In another of its aspects, the invention provides a method
for treatment of subjects to reduce gastrointestinal tissue mass as
part of regimen involving radiotherapy or chemotherapy, in which
there is administered to a subject a small bowel mass reducing
amount of the GLP-2 antagonist. The invention embraces both
co-administration of the GLP-2 antagonist with the radiotherapeutic
agent or the chemotherapeutic agent or alternatively administration
of the GLP-2 antagonist so as to reduce the growth of small bowel
tissue prior to initiation of the radiotherapy or chemotherapy.
Appropriate dosing regimens for GLP-2 antagonists may determined by
monitoring the subsequent reduction in intestinal damage and/or
recovery time after radiotherapy or chemotherapy.
[0071] In a further aspect, the invention provides a method for
treatment of obesity, which comprises administering to an obese
subject an effective amount of a GLP-2 antagonist capable of
antagonizing the effects of GLP-2, as evidenced by weight loss
under controlled dietary intake conditions.
[0072] In another aspect, the invention provides a method of
treatment of a subject suffering from a GLP-2 producing tumor which
comprises administering to a subject in need thereof an effective
amount of a GLP-2 antagonist capable of antagonizing the effects of
GLP-2, as evidenced by decrease in size of the intestinal
epithelium and small bowel mass over the treatment period.
[0073] Another use for antagonists of GLP-2 is as a therapy for
correcting a fluid imbalance due to a malabsorption problem across
the small intestine. The efficacy of the GLP-2 antagonist treatment
is monitored by assessing stool volumes ICF and ECF volume, urine
volume and osmolarity, blood pressure, and plasma electrolytes.
[0074] As used herein, the term "subject" includes a human or other
mammal, including livestock and pets.
[0075] F. Formulations of the GLP-2 Antagonists
[0076] For administration to subjects, including humans and
animals, the GLP-2 antagonists are provided, in one aspect of the
invention, in pharmaceutically or veterinarily acceptable form
(e.g., as a preparation that is sterile-filtered e.g. through a
0.22.mu. filter) and substantially pyrogen-free. Desirably, the
GLP-2 antagonist to be formulated migrates as a single or
individualized peak on HPLC, exhibits uniform and authentic amino
acid composition and sequence upon analysis thereof, and otherwise
meets standards set by the various national bodies which regulate
quality of pharmaceutical or veterinary products.
[0077] For therapeutic use, the chosen GLP-2 antagonist is
formulated with a carrier that is pharmaceutically or veterinarily
acceptable and is appropriate for delivering the peptide by the
chosen route of administration. Suitable pharmaceutically or
veterinarily acceptable carriers are those used conventionally with
peptide-based drugs, such as diluents, excipients and the like.
Reference may be made to "Remington's Pharmaceutical Sciences",
17th Ed., Mack Publishing Company, Easton, Pa., 1985, for guidance
on drug formulations generally. In one embodiment of the invention,
the compounds are formulated for administration by infusion or by
injection, either sub-cutaneously or intravenously, and are
accordingly utilized as aqueous solutions in sterile and
pyrogen-free form and optionally buffered to a slightly acidic or
physiological pH. Thus, the compounds may be administered in
distilled water or, more desirably, in saline, buffered saline or
5% dextrose solution. Water solubility may be enhanced, if desired,
by incorporating a solubility enhancer, such as acetic acid.
[0078] The aqueous carrier or vehicle can be supplemented for use
as injectables with an amount of gelatin that serves to depot the
GLP-2 antagonist at or near the site of injection, for its slow
release to the desired site of action. Concentrations of gelatin
effective to achieve the depot effect are expected to lie in the
range from 10-20%. Alternative gelling agents, such as hyaluronic
acid, may also be useful as depoting agents.
[0079] The GLP-2 antagonists of the invention may also be
formulated as a slow release implantation device for extended and
sustained administration of GLP-2 antagonist. Examples of such
sustained release formulations include composites of biocompatible
polymers, such as poly(lactic acid), poly(lactic-co-glycolic acid),
methylcellulose, hyaluronic acid, collagen, and the like. The
structure, selection and use of degradable polymers in drug
delivery vehicles have been reviewed in several publications,
including, A. Domb et al., Polymers for Advanced Technologies
3:279-292 (1992). Additional guidance in selecting and using
polymers in pharmaceutical or veterinary formulations can be found
in the text by M. Chasin and R. Langer (eds.), "Biodegradable
Polymers as Drug Delivery Systems," Vol. 45 of "Drugs and the
Pharmaceutical Sciences," M. Dekker, New York, 1990. Liposomes may
also be used to provide for the sustained release of a GLP-2
antagonist. Details concerning how to use and make liposomal
formulations of drugs of interest can be found in, among other
places, U.S. Pat. No. 4,944,948; U.S. Pat. No. 5,008,050; U.S. Pat.
No. 4,921,706; U.S. Pat. No. 4,927,637; U.S. Pat. No. 4,452,747;
U.S. Pat. No. 4,016,100; U.S. Pat. No. 4,311,712; U.S. Pat. No.
4,370,349; U.S. Pat. No. 4,372,949; U.S. Pat. No. 4,529,561; U.S.
Pat. No. 5,009,956; U.S. Pat. No. 4,725,442; U.S. Pat. No.
4,737,323; U.S. Pat. No. 4,920,016. Sustained release formulations
are of particular interest when it is desirable to provide a high
local concentration of a GLP-2 antagonist or prolonged circulating
levels.
[0080] The GLP-2 antagonist can be utilized in the form of a
sterile-filled vial or ampoule, that contains an amount of the
peptide effective to antagonize endogenous GLP-2 activity, in
either unit dose or multi-dose amounts. The vial or ampoule may
contain the GLP-2 antagonist and the desired carrier, as an
administration-ready formulation. Alternatively, the vial or
ampoule may contain the GLP-2 peptide in a form, such as a
lyophilized form, suitable for reconstitution in a suitable
carrier, such as phosphate-buffered saline.
[0081] As an alternative to injectable formulations, the GLP-2
antagonist may be formulated for administration by other routes.
Oral dosage forms, such as tablets, capsules and the like, can be
formulated in accordance with standard pharmaceutical or veterinary
practice.
[0082] Finally, chronic delivery of the GLP-2 antagonist for weight
loss or other therapeutic indications may be achieved through gene
therapy techniques. For example, cells may be engineered ex vivo to
express high levels of the GLP-2 antagonist, and then such cells
may be implanted into the subject animal or human for therapeutic
efficacy.
[0083] G. Dosages of the GLP-2 Antagonists of the Invention
[0084] The therapeutic dosing and regimen most appropriate for
treatment will of course vary with the disease or condition to be
treated, and according to the subject's weight and other
parameters. The results presented herein below demonstrate that a
dose of GLP-2 antagonist equivalent to about 1 mg/kg to 100
.mu.g/kg (or less) administered twice daily over 10 days can
generate very significant decrease in small bowel mass. It is
conceivable that much smaller doses, e.g., in the .mu.g/kg range,
and shorter or longer duration or frequency of treatment, will also
produce therapeutically useful results, i.e., a statistically
significant decrease particularly in small bowel mass. The dosage
sizes and dosing regimen most appropriate for human use are guided
by the results herein presented, and can be confirmed in properly
designed clinical trials.
[0085] An effective dosage and treatment protocol may be determined
by conventional means, starting with a low dose in laboratory
animals and then increasing the dosage while monitoring the
effects, and systematically varying the dosage regimen as well.
Numerous factors may be taken into consideration by a clinician
when determining an optimal dosage for a given subject. Primary
among these is the amount of GLP-2 normally circulating in the
plasma, which is on the order of 151 pmol/ml in the resting state,
rising to 225 pmol/ml after nutrient ingestion for healthy adult
humans (Orskov, C. and Holst, J. J., 1987, Scand. J. Clin. Lav.
Invest. 47:165). Additional factors include the size of the
subject, the age of the subject, the general condition of the
subject, the particular disease being treated, the severity of the
disease, the presence of other drugs in the subject, the in vivo
activity of the GLP-2 antagonist and the like. The trial dosages
would be chosen after consideration of the results of animal
studies and the clinical literature. It will be appreciated by the
person of ordinary skill in the art that information such as
binding constants and Ki derived from in vitro GLP-2 binding
competition assays may also be used in calculating dosages, as well
as the calculated half-life of the GLP-2 antagonist in vivo.
[0086] A typical human dose of a GLP-2 antagonist would be from
about 10 .mu.g/kg body weight/day to about 10 mg/kg/day, preferably
from about 50 .mu.g/kg/day to about 5 mg/kg/day, and most
preferably about 100 .mu.g/kg/day to 1 mg/kg/day. As it is
conceivable that the GLP-2 antagonists of the invention could be up
to 10 to even 100 times more potent than GLP-2, a typical dose of
such a GLP-2 antagonist may be lower, for example, from about 100
ng/kg body weight/day to 1 mg/kg/day, preferably 1 .mu.g/kg/day to
500 .mu.g/kg/day, and even more preferably 1 .mu.g/kg/day to 100
.mu.g/kg/day.
EXAMPLE 1
GLP-2 Antagonist Synthesis
[0087] The following GLP-2 antagonist peptides were
synthesized:
[0088] [Gly.sup.2, Ala.sup.15]GLP-2; [Gly.sup.2, Ala.sup.22]GLP-2;
[Gly.sup.2, Ala.sup.29]GLP-2; [Gly.sup.2, Ala.sup.32]GLP-2;
[Gly.sup.2, Ala.sup.33]GLP-2; ratGLP-2(2-33); ratGLP-2(3-33);
ratGLP-2(4-33); ratGLP-2(5-33); [Leu.sup.2]GLP-2; [Glu.sup.2]GLP-2;
[Arg.sup.2]GLP-2; [Trp.sup.2]GLP-2; [Cys.sup.2]GLP-2;
[PO.sub.3-Tyr.sup.2]GLP-2; and [Phg.sup.2]GLP-2.
[0089] Solid phase peptide synthesis (SPPS) was carried out
manually in a 300 milliliter (ml) vessel on a 3 millimole (mmole)
scale using 6 grams (g) of chloromethyl (Merrifield) resin (for
C-terminal free acid peptides) with a substitution of 0.5
milliequivalents (meq) per gram. Amino acids were protected at the
amino-terminus with the t-butyloxycarbonyl (tBoc) group. The
side-chains of trifunctional amino acids were protected with the
benzyl (Bz, for serine and threonine), benzyloxymethyl (BOM, for
histidine), 2-bromobenzyloxycarbonyl (2-BrZ, for tyrosine),
2-chlorobenzyloxycarbonyl (2-ClZ, for lysine), cyclohexyl (cHex,
for aspartic and glutamic acids), and tosyl (Tos, for arginine)
groups. The first amino acid was coupled to the chloromethyl resin
through esterification of the protected amino acid in the presence
of potassium fluoride (KF). C-terminal amide peptides were
synthesized on a 4-methylbenzhydrylamine (MBHA) resin on a 3 mmol
scale using 6 g of resin with a substitution of 0.5 meq/g. The
first amino acid was coupled to the MBHA resin according to the
procedure described for peptide elongation.
[0090] Amino-group deprotection was carried out using 50%
trifluoroacetic acid (TFA) in dichloromethane (CH.sub.2Cl.sub.2),
followed by neutralization using two washes of 10% triethylamine
(Et.sub.3N) in CH.sub.2Cl.sub.2. Peptide elongation was carried out
using N,N-dicyclohexylcarbodiimide/1-hydroxybenzotriazole
(DCC/HOBt) activation in CH.sub.2Cl.sub.2/dimethylformamide (DMF).
The growing peptide chain was capped after each elongation step
with 20% Ac.sub.2O in CH.sub.2Cl.sub.2. The peptide-resin was
washed after each elongation, capping and deprotection step with
isopropanol (iPrOH) and methanol (MeOH). The washes were repeated
once. N-terminal acetyl peptides were prepared by acetylation of
the terminal amino-group with 20% Ac.sub.2O in CH.sub.2Cl.sub.2
after deprotection and neutralization as described. Resin-bound
products were routinely cleaved by a low-high procedure using
hydrogen fluoride (HF) containing dimethylsulfide (DMS) and
p-cresol as scavengers.
[0091] Crude peptides were purified by preparative high pressure
liquid chromatography (HPLC) using a Vydac C18, 15-20 .mu.m
wide-pore, 2 inch.times.12 inch, reverse-phase silica column using
gradient elution with 0.1% TFA in water modified with acetonitrile.
Elution was monitored at 220 nanometers (nm). Each fraction
collected was analyzed for purity by analytical HPLC using a Vydac
C18, 5 .mu.m, 4.6.times.254 millimeter (mm), reverse-phase silica
column by gradient elution using 0.1% TFA in water modified with
acetonitrile, and monitored at 215 nm. Fractions demonstrating
greater than 95% purity were combined and lyophilized. Acetate
salts of the peptides were prepared from the TFA salts by
dissolution of the lyophilized powder in water, with addition of
acetonitrile to aid in dissolution when necessary. The solution was
passed through a protonated Bio-Rex-70 cation exchange resin. The
resin was washed with 5 bed-volumes of water, and the resin-bound
peptide eluted with 50% acetic acid in water. The eluent was
diluted with water and lyophilized.
[0092] The final lyophilized powder was analyzed for purity by two
analytical reverse-phase HPLC methods using a Vydac C18, 5 .mu.m,
4.6.times.254 mm reverse-phase silica column. Two solvent systems
were used: a gradient of water adjusted to pH 2.25 with
triethylamine phosphate, modified with acetonitrile; and a gradient
of 0.1% TFA in water, modified with acetonitrile. The column eluent
was monitored at 215 nm. The identity of each product was confirmed
by amino acid analysis and by electrocopy-mass spectroscopy.
[0093] The GLP-2 antagonists were next formulated as described
below in Example 2. Each of the GLP-2 antagonists was fully soluble
in water at room temperature unless otherwise noted.
EXAMPLE 2
GLP-2 Antagonist Formulation
[0094] The GLP-2 antagonists were formulated for injection either
in phosphate buffered saline or as a gelatin-containing depot
formulation. For the PBS-formulated GLP-2 antagonist preparations,
a 10.times. stock PBS solution was first prepared, using 80 g NaCl
(BDH ACS 783), 2g KCl (BDH ACS 645), 11.5 g Na.sub.2HPO.sub.4
(Anachemia AC-8460), and 2 g KH.sub.2PO.sub.4 (Malinckrodt AR7100),
which was brought to a total volume of one litre with sterile
distilled water. The final working solution was obtained by 10:1
dilution of the stock solution with sterile distilled water and
adjusted to pH 7.3-7.4 if necessary, using sufficient volumes of
10N Na OH. The working solution was then autoclaved for 30 minutes.
In the final working PBS solution, concentrations were 137 mM NaCl,
2.7 mM KC1, 4.3 mM Na.sub.2HPO.sub.4.7H2O , and 1.4 mM
KH.sub.2PO.sub.4.
[0095] The GLP-2 antagonists, as a powdered peptide, were added to
the working PBS solution as required to generate formulations
having the desired peptide concentrations. For example, to generate
a PBS solution of GLP-2 antagonist at 130 mg/l, 5.2 mg of GLP-2
antagonist was dissolved in 40 ml of PBS to yield a GLP-2
antagonist concentration of 130 .mu.g/ml, and filter sterilized.
0.5 ml of the GLP-2 antagonist solution was injected twice
daily.
[0096] To generate the gelatin-based GLP-2 antagonist formulations,
a gelatin solution was first prepared by dissolving 12 grams of
gelatin (Sigma, G-8150 Lot #54HO7241 Type A from Porcine skin
[9000-70-8]-300 Bloom) in 100 ml distilled water. The gelatin
solution was then autoclaved, warmed at 37.degree. C., and the
GLP-2 antagonist previously dissolved in phosphate buffered saline
as described above was added to achieve specific, desired peptide
concentrations. For instance, to generate a gelatin-based PBS
solution of the GLP-2 antagonist at a concentration of 130 mg/l, 10
ml of a PBS solution prepared with 5.2 mg of GLP-2 antagonist was
diluted with 30 ml of the 20% working gelatin solution as first
described above. The solution was mixed by gentle pipeting, to
yield a final solution of 130 mg/l GLP-2 antagonist in PBS/15%
gelatin.
EXAMPLE 3
Assay for Resistance to Dipeptidyl Peptidase IV
[0097] The following peptides were tested for resistance to
dipeptidyl peptidase IV (DPP-IV): a control peptide, ratGLP-2; the
[D-Ala.sup.2]ratGLP-2 agonist; and the [Gly.sup.2]ratGLP-2 agonist.
Additionally, the following peptides were also tested for DPP-IV
resistance: [Gly.sup.2, Ala.sup.15]GLP-2, [Gly.sup.2,
Ala.sup.22]GLP-2, [Gly.sup.2, Ala.sup.29]GLP-2, [Gly.sup.2,
Ala.sup.32]GLP-2, [Gly.sup.2, Ala.sup.33]GLP-2, [Leu.sup.2]GLP-2,
[Glu.sup.2]GLP-2, [Arg.sup.2]GLP-2, [Trp.sup.2]GLP-2,
[PO.sub.3-Tyr.sup.2]GLP-2, and [Cys.sup.2]GLP-2. To perform the
assay, 2.5 microliters (.mu.l) of a solution of human placental
DPP-IV (Calbiochem, La Jolla, Calif., cat. #317624) containing
0.125 milliunits (mU) of enzyme in 50% glycerol, 10 mM Tris, pH
7.8, EDTA and 0.02% NaN.sub.3 was added to 50 .mu.l of a solution
of the test peptide prepared at a concentration of 0.2 mg/ml in PBS
at pH 7.4. The mixture was incubated at 37.degree. C. in a
circulating water bath for 24 hours. The incubation was quenched by
the addition of 50 .mu.l of a solution of diprotin A prepared at a
concentration of 4 mg/ml in PBS. Each peptide was tested in
duplicate.
[0098] Each sample was analyzed by reverse-phase (RP) HPLC as
follows: 90 .mu.l of the quenched incubation mixture was injected
onto a Rainin Dynamax 300 .ANG., C18, 5 micron, 4.6.times.250
millimeter column. The samples were eluted with 0.1%
trifluoroacetic acid (TFA) in water modified with 0.1% acetonitrile
using a linear gradient and a flow rate of 1 ml per minute. Sample
components were detected at 214 nanometers (nm). The extent of
cleavage was measured by relative integration of the peak
corresponding to the cleavage product compared to that of the
remaining undigested parent peptide. The cleavage product of the
control peptide, ratGLP-2(1-33), which should be ratGLP-2(3-33),
was confirmed to have resulted from cleavage between residues
Ala.sup.2 and Asp.sup.3 by comparison of the retention time of this
component to that of a synthetic peptide standard, ratGLP-2(3-33),
and by collection of the product from the HPLC and analysis by mass
spectrometry.
[0099] After the 24 hour incubation, 22% of the control peptide,
ratGLP-2, was cleaved by DPP-IV. No cleavage products were detected
for the peptides [D-Ala.sup.2]ratGLP-2, [Gly.sup.2]ratGLP-2,
[Gly.sup.2, Ala.sup.15]GLP-2, [Gly.sup.2, Ala.sup.22]GLP-2,
[Gly.sup.2, Ala.sup.29]GLP-2, [Gly.sup.2, Ala.sup.32]GLP-2,
[Gly.sup.2, Ala.sup.33]GLP-2, [Leu.sup.2]GLP-2, [Glu.sup.2]GLP-2,
[Arg.sup.2]GLP-2, [Trp.sup.2]GLP-2, [PO.sub.3-Tyr.sup.2]GLP-2, and
[Cys.sup.2]GLP-2 after 24 hours.
EXAMPLE 4
GLP-2 Antagonist Assessment by Administration into Mice
[0100] Recipients were CD1 mice obtained from Charles River
Laboratory (Ontario, Canada). The CD1 mice were aged-matched
females at time of injection (n=3-4 per group), 6 weeks of age,
unless otherwise specified. The animals were allowed a minimum of
24 hours to acclimatize to the laboratory facility before the
initiation of each experiment. Animals were identified by ear
punch. The mice were not restricted by diet or activity during the
experiments. The light/dark cycle was 12 hours, between 6 pm to 6
am. Controls were age- and sex-matched (n=3-4) animals. Mice were
injected subcutaneously, twice a day (b.i.d.), with 2.5 .mu.g
peptide in a total volume of 0.5 cc of PBS and were monitored daily
in the laboratory facility. Animals were sacrificed 10 or 14 days
after injection, and were fasted at least 20 hours before
sacrifice.
[0101] The mice were anaesthetized with CO.sub.2 and exsanguinated
by cardiac puncture. Blood was collected in 75 .mu.l of TED
(Trasysol; EDTA (5000 KIU/ml: 1.2 mg/ml; Diprotin-A), and the blood
was centrifuged at 14 k.times.g for 5 minutes and the plasma was
stored at -70 prior to analysis. The small bowel was removed from
the peritoneal cavity, from pylorus to cecum, cleaned weighed and
measured. For comparative purpose, sections from each animal were
obtained from the identical anatomical position. Fragments each
measuring 1.5-2.0 cm in length were obtained 8.+-.2 cm, 18.+-.2 cm,
32.+-.2 cm from pylorus for histomorphometry representing proximal
jejunum, distal jejunum and distal ileum. Each small bowel fragment
was opened longitudinally on its antimesenteric border in a tissue
block and then placed on 10% formalin (vol./vol.) overnight, then
transferred to 70% ETOH.
[0102] Percentage change in small bowel weight was calculated by
dividing the mean change in bowel weight of antagonist treated
mice, relative to mice treated with vehicle only, by the mean bowel
weight of mice treated with vehicle only, and multiplying this
figure by 100.
2 TABLE 1 % Decrease in Small Bowel GLP-2 antagonist Weight
[Gly.sup.2, Ala.sup.15]GLP-2 14 [Gly.sup.2, Ala.sup.22]GLP-2 8
[Gly.sup.2, Ala.sup.29]GLP-2 19 [Gly.sup.2, Ala.sup.32]GLP-2 17
[Gly.sup.2, Ala.sup.33]GLP-2 6 [Leu.sup.2]GLP-2 23 [Glu.sup.2]GLP-2
25 [Arg.sup.2]GLP-2 23 [Trp.sup.2]GLP-2 5 [Cys.sup.2]GLP-2 20
[PO.sub.3-Tyr.sup.2]GLP-2 6 [Phg.sup.2]GLP-2 2
[0103] These results establish that antagonists of human GLP-2
which contain substitutions of the conserved residues at positions
15, 29, 32, or 33 with an alanine residue will actually cause a
decrease in small bowel weight when injected into mice. In
contrast, analogs of human GLP-2 which contain a wild type residue
at these positions (but which do contain the Gly.sup.2
substitution) will increase small bowel weight when injected into
mice using an identical experimental protocol (data not shown).
Therefore, we conclude that substitution of the residues at
positions 15, 29, 32, or 33 partially disrupts GLP-2 functional
activity and results in a GLP-2 antagonist.
[0104] Additionally, this data also shows the extremely surprising
result that substitutions of the Ala.sup.2 position with an amino
acid residue other than Gly, specifically Leu, Glu, Arg, Trp, Cys,
PO.sub.3-Tyr, and Phg, resulted in antagonistic activity.
EXAMPLE 5
GLP-2 Antagonist Assessment by Co-administration into Mice with
GLP-2
[0105] The candidate peptide antagonist and rat GLP-2 were
dissolved in PBS to give a final ratio of 25 .mu.g antagonist/2.5
.mu.g GLP-2 per 0.5 ml phosphate buffered saline solution (for a
10:1 ratio), or 12.5 .mu.g antagonist/2.5 .mu.g GLP-2 per 0.5 ml
phosphate buffered saline solution (for a 4:1 ratio), as indicated.
The antagonist/GLP-2 mixture was administered to 6-8 week old CD1
female mice subcutaneously, with the amount of peptide injected
being 25 .mu.g antagonist/2.5 .mu.g GLP-2 in 0.5 ml twice a day, or
12.5 .mu.g antagonist/2.5 .mu.g GLP-2 in 0.5 ml twice a day. After
10-14 days, peptide-injected and control (saline-injected) mice
were sacrificed, and small bowel weights were determined.
3TABLE 2 Ratio of Antagonist to GLP-2 administered # GLP-2
antagonist % Antagonism [by Weight] 1 ratGLP-2(2-33) 42 10:1 2
ratGLP-2(3-33) 33 4:1 3 ratGLP-2(4-33) 32 10:1 4 ratGLP-2(5-33) 16
10:1
[0106] These results illustrate that deletions of the first one to
four residues of GLP-2 results in an antagonist which will
antagonize the intestinotrophic activity of rat GLP-2 when
co-injected into experimental mice. These results are significant
for at least two reasons. First, this data reveals that the extreme
amino terminus of the GLP-2 peptides is involved in the
intestinotrophic effect of GLP-2. Therefore, other alterations
which disrupt this terminus, for example, substitutions of amino
acids with opposite properties, instead of deletions, will likely
also convey antagonistic activity to the resulting analog. Second,
co-administration of antagonist and GLP-2 will serve to decrease or
even eliminate the intestinotrophic effect of GLP-2. Antagonists of
GLP-2 may therefore be administered to a subject in situations
where excess production of GLP-2 occurs, for example, a subject
with a tumor which secretes GLP-2 and/or responds trophically to
GLP-2 peptide.
INCORPORATION BY REFERENCE
[0107] All patents, patent applications, and publications cited are
incorporated herein by reference.
EQUIVALENTS
[0108] The foregoing written specification is sufficient to enable
one skilled in the art to practice the invention. Indeed, various
modifications of the above-described means for carrying out the
invention which are obvious to those skilled in the field of
molecular biology, protein chemistry, medicine or related fields
are intended to be within the scope of the following claims.
Sequence CWU 1
1
1 1 33 PRT Homo sapiens 1 His Ala Asp Gly Ser Phe Ser Asp Glu Met
Asn Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn
Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp
* * * * *